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Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS

Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher...

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Autores principales: Sloth, Ane Beth, Bakhshinejad, Babak, Stavnsbjerg, Camilla, Rossing, Maria, Kjaer, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10049078/
https://www.ncbi.nlm.nih.gov/pubmed/36982469
http://dx.doi.org/10.3390/ijms24065396
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author Sloth, Ane Beth
Bakhshinejad, Babak
Stavnsbjerg, Camilla
Rossing, Maria
Kjaer, Andreas
author_facet Sloth, Ane Beth
Bakhshinejad, Babak
Stavnsbjerg, Camilla
Rossing, Maria
Kjaer, Andreas
author_sort Sloth, Ane Beth
collection PubMed
description Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.(TM)-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries.
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spelling pubmed-100490782023-03-29 Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS Sloth, Ane Beth Bakhshinejad, Babak Stavnsbjerg, Camilla Rossing, Maria Kjaer, Andreas Int J Mol Sci Article Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.(TM)-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries. MDPI 2023-03-11 /pmc/articles/PMC10049078/ /pubmed/36982469 http://dx.doi.org/10.3390/ijms24065396 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sloth, Ane Beth
Bakhshinejad, Babak
Stavnsbjerg, Camilla
Rossing, Maria
Kjaer, Andreas
Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
title Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
title_full Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
title_fullStr Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
title_full_unstemmed Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
title_short Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
title_sort depth of sequencing plays a determining role in the characterization of phage display peptide libraries by ngs
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10049078/
https://www.ncbi.nlm.nih.gov/pubmed/36982469
http://dx.doi.org/10.3390/ijms24065396
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